Broadband laser illumination device for a scanning microscope with output stabilization

ABSTRACT

The arrangement for examining microscope preparations with a scanning microscope comprises a laser ( 1 ) and an optical means ( 12 ) which images the light generated by the laser ( 1 ) onto a specimen ( 13 ) that is to be examined. Provided between the laser ( 1 ) and the optical means ( 12 ) is an optical component ( 3, 20 ) that spectrally spreads, with a single pass, the light generated by the laser ( 1 ). The optical component ( 3, 20 ) is made of photonic band-gap material. It is particularly advantageous if the photonic band-gap material is configured as a light-guiding fiber ( 20 ).

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.11/580,065, filed Oct. 13, 2006, which is a continuation of U.S.application Ser. No. 11/034,888, filed Jan. 14, 2005, which is adivisional of U.S. application Ser. No. 09/881,062, filed Jun. 15, 2001,which claims priority of German Patent Application Nos. 100 30 013.8,filed Jun. 17, 2000 and 101 15 509.3, filed Mar. 29, 2001, the entirecontents of each application is incorporated herein by reference.

FIELD OF THE INVENTION

The invention concerns an arrangement for examining microscopepreparations with a scanning microscope, In particular, the inventionconcerns an arrangement for examining microscopic preparations with ascanning microscope that comprises a laser and an optical means whichimages the light generated by the laser onto a specimen that is to beexamined. The scanning microscope can also be configured as a confocalmicroscope.

The invention furthermore concerns an illumination device for a scanningmicroscope.

BACKGROUND OF THE INVENTION

In scanning microscopy, a specimen is scanned with a light beam. Lasersare often used as the light source for this purpose. An arrangementhaving a single laser which emits several laser lines is known, forexample, from EP 0 495 930, “Confocal microscope system for multi-colorfluorescence.” Mixed-gas lasers, in particular ArKr lasers, are usuallyused at present for this purpose.

Diode lasers and solid-state lasers are also in use.

U.S. Pat. No. 5,161,053 entitled “Confocal microscope” discloses aconfocal microscope in which light of an external light source istransported with the aid of a glass fiber to the beam path of themicroscope and the end of the glass fiber serves as a point lightsource, so that a mechanical stop is superfluous.

The emission spectrum of lasers is confined to a narrow wavelengthrange, so that for simultaneous multiple-line excitation, the light ofseveral lasers must be combined into one illumination beam.

The gas lasers usually used as multiple-line lasers are very complex andexpensive. They moreover require a great deal of maintenance, makingthem difficult to use continuously in many microscopy applications.

SUMMARY OF THE INVENTION

It is the object of the invention to create a scanning microscope whichmakes possible specimen examination with several spectral lines withoutrequiring the use of multiple-line lasers or more than one laser.

The aforesaid object is achieved by a scanning microscope comprising: alaser, an optical means for imaging light generated by the laser onto aspecimen and an optical component positioned between the laser and theoptical means, wherein the light generated by the laser passes throughthe optical component whereby the optical component spectrally spreadsthe light passing through.

A further object of the invention is to create an illumination devicefor a scanning microscope which provides an illumination encompassing anumerous selectable spectral regions.

The aforesaid object is achieved by an illumination device comprising alaser which has a light exit opening, an optical component made ofphotonic band-gap material which is mounted at the light exit opening.

It a further object of the invention to create a confocal scanningmicroscope which makes possible specimen examination with severalspectral lines without requiring the use of multiple-line lasers or morethan one laser.

The aforesaid object is achieved by a confocal scanning microscopecomprising: a laser, an optical means for imaging light generated by thelaser onto a specimen, a detector for receiving light coming from thespecimen, an optical component positioned between the laser and theoptical means, wherein the light generated by the laser passes throughthe optical component, whereby the optical component spectrally spreadsthe light passing through and an illumination pinhole through which thespecimen is illuminated by the light emerging from the opticalcomponent.

It a further object of the invention to create a scanning microscopewhich makes possible specimen examination with several spectral lineswithout requiring the use of multiple-line lasers or more than one laserand which is realized in a simple and cost effective way.

The aforesaid object is achieved by a scanning microscope comprising: apulsed laser, an optical means for imaging light generated by the pulsedlaser onto a specimen and a tapered light-guiding fiber positionedbetween the pulsed laser and the optical means, wherein the lightgenerated by the pulsed laser passes through the tapered light-guidingfiber whereby the tapered light-guiding fiber spectrally spreads thelight passing through.

The optical component in the form of a photonic band-gap material hasthe advantage that the optically nonlinear construction of the fibercauses a short laser pulse to be spread out, thus creating a spectrallybroad, continuous light spectrum.

A “photonic band-gap material” is a microstructured, transparentmaterial. It is possible, usually by assembling various dielectrics, toimpart to the resulting crystal a band structure which is reminiscent ofthe electron band structure of semiconductors.

The technology has recently also been implemented in light-guidingfibers. The fibers are manufactured by drawing out structured glasstubes. The fibers have a particular underlying structure: smallcapillaries are left open in the fiber direction, spaced approximately2-3 μm apart and with a diameter of approx. 1 μm, and usually filledwith air. No capillaries are present in the center of the fiber. Thesekinds of fibers are known as “photon crystal fibers,” “holey fibers,” or“microstructured fibers.”

Photon crystal fibers can be used to produce a continuous spectraldistribution over the entire visible wavelength region. This is done bycoupling the light of a short-pulse laser into the fiber. The opticallynonlinear construction of the fiber causes the frequency spectrum of thelaser to spread out, creating a spectrally broad, continuous spectrum.

It is an other advantage of the invention to provide an embodiment whichis simple an cost effective to realize. The optical component is alight-guiding fiber with a fiber core, wherein the fiber has a thinningprovided on a part of the fiber. Light-guiding fibers of that kind areknown as “tapered fibers”. Preferable, the light-guiding fiber has anoverall length of one meter an the thinning is provided over a length of30 mm to 90 mm. The diameter of the fiber is 150 μm and diameter of thefiber core is approx. 8 μm. A the thinning the diameter of the fiber isreduced to approx. 2 μm. Consequently the diameter of the fiber core isthe range of a few nanometers.

For use in microscopy, it is important to implement means for wavelengthselection and for light output stabilization. A fiber laser of this kindcan therefore advantageously be combined with acoustooptical orelectrooptical tunable filters (AOTFs), acoustooptical or electroopticaldeflectors (AODs), or acoustooptical or electrooptical beam splitters(AOBSs). These can be used not only for wavelength selection but also toblock out detected light (our German application DE 199 06 757 A1:“Optical arrangement”).

In confocal microscopy in particular, the fiber exit end can be used asa point light source, thus making the use of an excitation aperturesuperfluous. With a configuration of this kind, it would be particularlyadvantageous for the fiber end itself to have a partially reflectivecoating, so that this partial reflector forms a resonator end mirror.

Further embodiments make provision for apparatuses to compensate forlight output fluctuations. It is possible, for example, to incorporate acontrol loop for light output stabilization, which measures the lightoutput in the beam path of the microscope in parasitic fashion, andmaintains a constant specimen illumination light output by, for example,varying the pumping light output or with the aid of an acoustooptical orelectrooptical element. LCD attenuators could also be used for thispurpose.

A further advantage of the invention is that if the illumination deviceis already appropriately configured, it supplies several spectralregions for illumination. The laser which constitutes the illuminationdevice for a scanning microscope has an optical component attached atthe light exit opening. The optical component is made of photonicband-gap material. The photonic band-gap material can also be configuredas a light-guiding fiber.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter of the invention is schematically depicted in thedrawings and is described below with reference to the Figures, in which:

FIG. 1 shows an arrangement according to the present invention with aconfocal microscope;

FIG. 2 shows an arrangement in which an illumination pinhole has beenomitted,

FIG. 3 shows an arrangement with light output stabilization,

FIG. 4 shows an embodiment of the optical component and

FIG. 5 shows a further embodiment of the optical component.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows a confocal microscope that uses an optical component 3 tospread out a laser pulse generated by a pulsed laser 1. Pulsed laser 1defines a pulsed laser beam 2 that is directed through optical component3. Optical component 3 is a photonic band-gap material. What emergesfrom optical component 3 is a spectrally broad-band illuminating light 4that is imaged by a first optical system 5 onto an illumination pinhole6 and then strikes a beam splitter 7. From beam splitter 7, thespectrally broad-band illuminating light 4 passes to a second opticalsystem 8 which generates a parallel light beam 4 a that strikes ascanning mirror 9. Scanning mirror 9 is followed by several opticalsystems 10 and 11 which shape light beam 4 a. Light beam 4 a passes toan objective 12, by which it is imaged onto a specimen 13. The lightreflected or emitted from the specimen defines an observation beam path4 b. The light of observation beam path 4 b passes once again throughsecond optical system 8, and is imaged onto a detection pinhole 14 thatsits in front of a detector 15. Optical component 3 makes it possible togenerate the laser light necessary for the examination of specimen 13 inaccordance with the desired spectrum.

The exemplary embodiment depicted in FIG. 2 shows a confocal microscopein which illumination pinhole 6 has been omitted. All elements identicalto the elements of FIG. 1 are labeled with the same referencecharacters. In this exemplary embodiment, an acoustooptical tunablefilter (AOTF) 16, which is connected to a corresponding AOTF drivesystem 17, is used instead of first optical system 5. Since opticalcomponent 3 can generate a broad-band illuminating light 4, it isnecessary to provide means for wavelength selection and for light outputstabilization. Advantageously, acoustooptical or electrooptical tunablefilters (AOTFs) can be combined with acoustooptical or electroopticaldeflectors (AODs) and acoustooptical or electrooptical beam splitters(AOBSs).

These can be used not only for wavelength selection but also to blockout detected light. Also associated with AOTF 16 is a beam dump 18 whichintercepts the unused spectral portions of the illuminating light inorder to prevent unnecessary disturbance of the scanning microscope.

A further embodiment of the invention is depicted in FIG. 3. Here alight-guiding fiber 20 made of the photonic band-gap material is usedinstead of optical component 3. From pulsed laser 1, pulsed laser beam 2is coupled via an optical system 19 into an entrance end 20 a oflight-guiding fiber 20. Since light-guiding fiber 20 is constructed fromthe photonic band-gap material, a spectrally spread laser pulse emergesfrom exit end 20 b and is coupled out via an optical system 21. Beforethe spectrally spread laser pulse strikes illumination pinhole 6,spectral filtering is performed. For that purpose, several color filters24 are arranged on a turret 23. Turret 23 can be rotated by a motor 22,so that the corresponding color filters 24 can be introduced into thebeam path. Also conceivable is a linear arrangement of color filters 24,in which case color filters 24 are moved by means of a linear motioninto an illumination beam path 50. After illumination pinhole 6,illumination beam path 50 is comparable to the beam path of FIG. 1. Asalready mentioned in FIG. 1, beam splitter 7 deflects the light ontoscanning mirror 9. A portion of the light passes through beam splitter 7and defines a lost beam path 50 a. This portion of the light is lost forobservation or measurement purposes. For this reason, there is providedin lost beam path 50 a a detector 25 which determines the lost light andascertains therefrom an electronic variable that is conveyed via a line30 to an electronic control system 26. Electronic control system 26 isconnected via a further line 32 to pulsed laser 1. Electronic controlsystem 26 regulates the intensity of pulsed laser 1, via line 32, insuch a way that a constant light output always strikes specimen 13. Forexample, a control loop can be provided for light output stabilization,in such way that it measures the light output in the beam path of themicroscope in parasitic fashion, and maintains a constant specimenillumination light output by, for example, varying the pumping lightoutput or with the aid of an acoustooptical or electrooptical element.LCD attenuators could also be used for this purpose.

FIG. 4 shows a schematic representation of the optical component 3. Theoptical component 3 is a conventional light-guiding fiber 51, which hasa overall diameter of 125 μm and the fiber core 52 has a diameter of 6μm. In the area of a thinning 53, which is approx. 300 mm long, theoverall diameter of the light-guiding fiber 51 is reduced 1.8 μm. Inthis area the diameter of the fiber core 52 is in the range of a fewnanometers.

FIG. 5 shows a further embodiment of the optical component 3. Theoptical component 3 is a microstructured optical element. It consists ofphotonic band gap material, which has a special honeycombedmicrostructure 54. The honeycombed structure 54 that is shown isparticularly suitable for generating broadband light. The diameter ofthe glass inner cannula 55 is approximately 1.9 μm. The inner cannula 55is surrounded by glass webs 56. The glass webs 56 form honeycombedcavities 57. These micro-optical structure elements together form asecond region 58, which is enclosed by a first region 59 that isdesigned as a glass cladding.

The present invention was described with reference to particularembodiments. It is self-evident, however, that changes and modificationscan be made without leaving the spirit and the scope of the claims.

1. An optical system for use in microscopy comprising: a laserconfigured to generate light having a wavelength range; imaging opticsconfigured to image light generated by the laser onto an image plane; anoptical component positioned between the laser and the imaging optics,wherein the light generated by the laser passes through the opticalcomponent, and wherein the optical component is configured to increasesaid wavelength range of said light to a substantial portion of theentire visible wavelength range; an attenuator for attenuating at leasta portion of at least one wavelength of light emerging from the opticalcomponent, and wherein the attenuator comprises at least one of aspectrally selective filter, an acoustooptical tuneable filter (AOTF),an acoustooptical deflector (AOD) and an LCD, and wherein the lasercomprises a pulsed laser; and a control loop for light outputstabilization, which measures the light output in the beam path of theimaging optics and which maintains a constant specimen illuminationlight output by varying pumping light output of the pulsed laser or withthe aid of an acoustooptical or electrooptical element.
 2. An opticalsystem as defined in claim 1, wherein the optical component comprisesphotonic band-gap material.
 3. An optical system as defined in claim 2,wherein the photonic band-gap material is configured as a light-guidingfiber.
 4. An optical system as defined in claim 1, wherein the opticalcomponent comprises a tapered light-guiding fiber.
 5. An optical systemas defined in claim 1, wherein the system comprises a microscope.